LCAT is an enzyme which acts on lecithin and free cholesterol as the substrates in blood to produce lysolecithin and cholesterol ester, said enzyme being biosynthesized in liver. Assay of LCAT is useful for liver function test and is also useful for pathologic study of lipoids metabolism disorder and diabetes mellitus, since activity of LCAT has a relation to disorder of liver cells. For example, LCAT is decreased in the cases of acute hepatitis, liver cirrhosis, closed jaundice and cancer of the liver, while increased in the cases of alcoholic liver disease and fatty liver.
Hitherto, assay of this enzyme has been carried out by determining change of an amount of free cholesterol consumed as substrate. Practically, it is conducted by warming blood serum (or blood plasma) for a definite period of time (e.g. 2 hours) at an optimal reaction temperature of 37.degree. C and determining change of an amount (n mole) of free cholesterol before and after the warming. The activity is indicated as a changed amount of n mole per ml of blood serum per hour (h), i.e., n mole/(ml.multidot.h).
Reaction rate of enzymes is usually indicated by that in the initial stage of a reaction where an enough amount of substrate exists in a reaction solution and reaction curve is linear relative to time. However, in the case of LCAT, assay is very difficult because the reaction occured in high density lipoprotein, apoprotein A-1 in high density lipoprotein acts as an activating factor for the enzyme and furthermore lecithin and cholesterol are essentially insoluble in water. Assay methods are divided into two main classes namely, the common substrate method where an excessive amount of substrate having no LCAT activity is added to a small amount of blood serum (or blood plasma), and the self -substrate method where the blood serum (or blood plasma) is warmed directly and endogenous lipoprotein is used as a substrate. In the latter case, it is difficult to determine change of amount of free cholesterol by spectrophotometrical analysis unless the warming time is made long, since reactivity of LCAT itself is essentially very small and linearlity of parameters is lost within one hour after initiation of the reaction when serum (or plasma) itself is warmed at 37.degree. C.
In order to dissolve difficulties mentioned above, there are many proposals. One approach is that lecithin comprising a surfactant is added to a specimen (Japanese Unexamined Patent Publication No. Sho 51-129293). This method is not satisfactory yet from a view point of clinical inspection, because the absence of free cholesterol, another substrate for LCAT, lowers the LCAT activity, and the surfactant added to solubilization of lecithin inhibits the LCAT activity.
Another approach is a treatment of a sample solution containing lecithin and free cholesterol as substrates treated by sonication in order to make it transparent and solubilized [Clinical Chemistry, Vol. 4, collected No. 3-4, 306-311, (1976)]. However, this approach is hardly applicable to clinical inspection, because the ultrasonic treatment only temporarily reduces turbidity of the solution but does not make it transparent and precipitation is produced after 2-3 hours.
The other approaches are those wherein incubation time of a specimen is reduced by keeping linearity of LCAT reaction or by increasing the activity thereof. For example, they include an assay of LCAT activity wherein polyanionic sugar derivatives are added to make cholesterol and lecithin transparent (Japanese Unexamined Patent Publication No. Sho 60-262600), and an assay of LCAT activity wherein a liposome comprising cholesterol and lecithin is added (referred to as "liposome method") [the 26th meeting of the Society of Clinical Chemistry held on Nov. 22, 1986, (Gist of the report, pp 100-101, 1986)]. However, these methods are not satisfactory yet, either, as enzyme, assay, since reaction time (incubation time) is longer than 40 minutes for the liposome method, and at least 1 hour for the other methods. Furthermore, these assays encounter difficulty in practical clinical inspection, since rapid assay is hardly effected. Even in the liposome method which requires the shortest period of time nowadays, it is very difficult to prepare homogeneous, stable liposome, and thus, it is difficult, indeed, to always supply liposome of the definite composition to the site of clinical diagnoses in general.
Recently, an assay for LCAT of exogenic lecithin and cholesterol esterase (Japanese Unexamined Patent Publication No. Sho 61-293396). This method involves also drawbacks in that it requires reaction time of longer than one hour to measure the increased amount of lysolecithin. As above mentioned, such a method is not desirable from a point of rapid measurement. Such a long reaction time owes to the limit of sensitivity of colorimetry.